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Position：Cell Counting Kit-8
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Q&A:
Cell Counting Kit-8
Product number CK101-01-100T CK101-01-500T
specification 100T 500T
specification 100T 500T
INTRODUCTION:
1) Inoculate cell suspension (100μL /well) in a 96-well plate. Also prepare wells that contain known numbers of viable cells (to create a calibration curve in step 5). Pre-incubate the plate in a humidified incubator (e.g., at 37 °C, 5% CO2 ).
Cell Counting Kit-8 (CCK-8) utilizes the highly water-soluble tetrazolium salt [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,monosodium salt] to produce a water-soluble formazan dye upon reduction in the presence of an electron carrier.
Cell Counting Kit-8 is a one-bottle solution; no premixing of components is required. Cell Counting Kit-8, being nonradioactive, allows sensitive colorimetric assays for the determination of the number of viable cells in cell proliferation and cytotoxicity assays. CCK is reduced by dehydrogenases in cells to give a yellow colored product (formazan), which is soluble in the tissue culture medium. The amount of the formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells. The detection sensitivity of CCK-8 is higher than other tetrazolium salts such as MTT, XTT, MTS or WST-1.
ADVANTAGES:
PROTOCOL:
1. Cell Proliferation Assay:
ADVANTAGES:
▷-One-bottle, ready-to-use solution
▷No organic solvents or isotopes required
▷No harvesting, no washing and no solubilization steps
▷More sensitive than MTT, XTT, MTS or WST-1
▷-Low toxicity.
STORAGE:
CCK-8 is stable for 2 years at -20 °C, 1 year at 4 °C and 6 months at room temperature with protection from light. Repeated thawing and freezing causes an increase in the background, which interferes with the assay. To avoid repeated thawing and freezing, keep the kit at 4 °C if it is frequently used.STORAGE:
PROTOCOL:
1. Cell Proliferation Assay:
2) Thaw the CCK-8 on the bench top or in a water bath at 37 °C if it is frozen. It takes about 30 minutes on the bench top at 25 °C or 5 minutes in a water bath at 37 °C.
3) Add 10μL of the CCK-8 solution to each well of the plate. Be careful not to introduce bubbles to the wells, since they interfere with the O.D. reading.
4) Incubate the plate for 1-4 hours in the incubator.
5) Measure the absorbance at 450 nm using a microplate reader. Prepare a calibration curve using the data obtained from the wells that contain known numbers of viable cells. To measure the absorbance later, add 10μL of 1% w/v SDS to each well, cover the plate and store it with protection from light at room temperature. No absorbance change should be observed for 48 hours.
2. Cytotoxicity Assay：
2. Cytotoxicity Assay：
1) Dispense 100 μ l of cell suspension (5000 cells/ well) in a 96-well plate.
2) Pre-incubate the plate for 24 hours in a humidified incubator (e.g., at 37 °C, 5% CO2 ).
3) Add 10 μL of various concentrations of toxicant into the culture media in the plate.
4) Incubate the plate for an appropriate length of time (e.g., 6, 12, 24 or 48 hours) in the incubator.
5) Thaw the CCK-8 on the bench top or in a water bath at 37 °C if it is frozen. It takes about 30 minutes on the bench top at 25 °C or 5 minutes in the water bath at 37 °C.
6) Add 10μL of CCK-8 solution to each well of the plate. Be careful not to introduce bubbles to the wells, since they interfere with the O.D. reading.
7) Incubate the plate for 1-4 hours in the incubator. Measure the absorbance at 450 nm using a microplate reader. To measure the absorbance later, add 10μL of 1% w/v SDS to each well, cover the plate and store it with protection from light at room temperature. No absorbance change should be observed for 48 hours.
TIPS:
TIPS:
1. Since the CCK-8 assay is based on the dehydrogenase activity detection in viable cells, conditions or chemicals that affect dehydrogenase activity in viable cells may cause discrepancy between the actual viable cell number and the cell number determined using the CCK-8 assay.
2. CCK may react with reducing agents to generate CCK formazan. Please check the background O.D. if reducing agents are used in cytotoxicity assays or cell proliferation assays.
3. Be careful not to introduce bubbles to the wells, since they interfere with the O.D. reading.
4. Phenol red containing culture media can be used with this kit for cell viability assays.
5. Membrane filtration is recommended for the sterilization of the CCK-8 solution, if necessary.
6. The incubation time varies by the type and number of cells in a well. Generally, leukocytes give weak coloration, thus a long incubation time (up to 4 hours) or a large number of cells (~105 cells/well) may be necessary.
7. Since the cytotoxicity of this kit is very low, further color development is possible after reading the absorbance.
8. Neutral red or crystal violet can be used after the CCK-8 assay.
9. Measure the reference wavelength at 600 nm or higher if there is a high turbidity in the cell suspension.
Q&A:
1. How many cells should there be in a well?
For adhesive cells, at least 1000 cells are necessary per well (100 μl medium) when using the kit's standard 96-well plate. For leukocytes, at least 2500 cells are necessary per well (100 μl medium) because of low sensitivity. The recommended maximum number of cells per well for the 96-well plate is 25000. If a 24-well or 6-well plate is used for this assay, please calculate the number of cells per well accordingly, and adjust the volume of the CCK-8 solution in a well to 10% of the total volume.
2. Does CCK-8 stain viable cells?
No, it does not stain viable cells because the water-soluble tetrazolium salt is used in the CCK-8 solution. The electron mediator, 1-Methoxy PMS, receives electrons from a viable cell and transfers the electron to CCK-8 in the culture medium. Since its formazan dye is also highly water-soluble, CCK-8 cannot be utilized for cell staining purpose.
3. How stable is CCK-8?
CCK-8 is stable for 2 years at -20 ℃, 1 year at 4 ℃, and 6 months at room temperature. CCK-8 is stable over 2 days even at 60℃ as long as the CCK-8 solution keeps its original red color and does not turn orange.
4. Does phenol red affect the assay?
No. The absorption value of phenol red in a culture medium can be removed by subtracting the absorption value of a blank solution from the absorption value of each well. Therefore, a phenol red containing mediumis usable for the CCK-8 assay.
5. Is there a correlation between CCK-8 and the Thymidine incorporation assay?
Yes. For correlation graphs, see page 1. Please note that since CCK-8 uses a different assaying mechanism from that of the Thymidine assay, the CCK-8 and Thymidine assay results may differ.
6. Is CCK-8 toxic to cells?
The toxicity of CCK-8 is so low that, after the CCK-8 assay is completed, the same cells can be used for other cell proliferation assays such as the crystal violet assay, neutral red assay or DNA fluorometric assay
7. I do not have a 450 nm filter. What other filters can I use?
You can use filters with the absorbance between 450 and 490 nm, even though 450 nm filter gives the best sensitivity